DESCRIPTION: The long-range objective of this research program is to understand in molecular terms how the centromere/kinetochore functions to bring about proper chromosome segregation during eukaryotic mitotic and meiotic cell divisions. Research in this area is highly relevant to human health problems, such as cancer and genetic defects due to chromosome imbalances. Cancer therapy often involves treatment with drugs that interfere with normal chromosome segregation. The research supported by this grant employs combined molecular and genetic strategies to elucidate the mechanics and regulation of centromere/kinetochore function in the budding yeast Saccharomyces cerevisiae. The functional kinetochore in budding yeast is comparatively simple (125 bp of CEN DNA plus five known associated proteins), and each kinetochore binds to only one microtubule on the mitotic spindle. Thus, the yeast centromere is an excellent experimental model to analyze the molecular mechanisms involved in eukaryotic kinetochore function. In previous research supported by this grant, we isolated a multisubunit protein complex (CBF3) that binds specifically to the CEN DNA. Affinity-purified CBF3 contains a minus-end-directed, microtubule-based motor activity (Kar3p), and this complex can link and move CEN DNA on microtubules. The specific aims of this project include 1) identification in vivo and in vitro of proteins interacting with the core kinetochore protein complex (CBF3); 2) further characterization of yeast kinetochore-microtubule interactions in vitro, including cell cycle-dependent regulation of the CBF3-Kar3p-microtubule interaction, and a study of the effects of CBF3-Kar3p on microtubule assembly-disassembly dynamics; 3) mechanistic studies on centromere/kinetochore proteins, including definition of a putative kinetochore-bound protein kinase (Cbf2p/Ndc10p) that is likely involved in regulation of kinetochore function; and 4) isolation and characterization of homologs of the essential yeast kinetochore proteins from higher eukaryotes, as a route to extend these studies to human biology.